NDLI: Generation of Genetically Stable Recombinant Rotaviruses Containing Novel Genome Rearrangements and Heterologous Sequences by Reverse Genetics

Content Provider	PubMed Central
Author	Navarro, Aitor Trask, Shane D. Patton, John T.
Copyright Year	2013
Abstract	The rotavirus (RV) genome consists of 11 segments of double-stranded RNA (dsRNA). Typically, each segment contains 5′ and 3′ untranslated regions (UTRs) that flank an open reading frame (ORF) encoding a single protein. RV variants with segments of atypical size owing to sequence rearrangements have been described. In many cases, the rearrangement originates from a partial head-to-tail sequence duplication that initiates after the stop codon of the ORF, leaving the protein product of the segment unaffected. To probe the limits of the RV genome to accommodate additional genetic sequence, we used reverse genetics to insert duplications (analogous to synthetic rearrangements) and heterologous sequences into the 3′ UTR of the segment encoding NSP2 (gene 8). The approach allowed the recovery of recombinant RVs that contained sequence duplications (up to 200 bp) and heterologous sequences, including those for FLAG, the hepatitis C virus E2 epitope, and the internal ribosome entry site of cricket paralysis virus. The recombinant RVs grew to high titer (>107 PFU/ml) and remained genetically stable during serial passage. Despite their longer 3′ UTRs, rearranged RNAs of recombinant RVs expressed wild-type levels of protein in vivo. Competitive growth experiments indicated that, unlike RV segments with naturally occurring sequence duplications, genetically engineered segments were less efficiently packaged into progeny viruses. Thus, features of naturally occurring rearranged segments, other than their increased length, contribute to their enhanced packaging phenotype. Our results define strategies for developing recombinant RVs as expression vectors, potentially leading to next-generation RV vaccines that induce protection against other infectious agents.
Related Links	http://dx.doi.org/10.1128/jvi.00413-13
Ending Page	6220
Page Count	10
Starting Page	6211
File Format	PDF
ISSN	0022538X
e-ISSN	10985514
Journal	Journal of Virology
Issue Number	11
Volume Number	87
Language	English
Publisher	American Society for Microbiology
Publisher Date	2013-06-01
Access Restriction	Open
Rights Holder	American Society for Microbiology
Subject Keyword	Immunology Virology Research in Higher Education
Content Type	Text
Resource Type	Article
Subject	Virology Immunology Microbiology Insect Science

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